Fluorescent proteins from aquatic species

Abstract

Provided are four new fluorescent proteins. The proteins were derived from two wild-type fluorescent proteins: a red fluorescent protein (RFP) that was isolated from Actinodiscus orDiscosoma sp. 1 and a green fluorescent protein (GFP) isolated from Montastraea cavernosa. Two mutant forms were generated from each wild-type protein. Each of the mutated forms has a higher fluorescence intensity than the respective wild-type form. The mutant forms of the fluorescent proteins allow for more sensitive detection of the fluorescence emitted by the proteins. Additionally, one of the mutant proteins is more resistant to photobleaching than its wild-type protein. The invention also encompasses isolated nucleic acids encoding the mutant forms of the wild-type RFP and GFP.

Description

CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a divisional of U.S. patent application Ser. No. 10 / 314,936, filed Dec. 9, 2002, which is incorporated herein by reference in its entirety.REFERENCE TO GOVERNMENT GRANT [0002] This invention was made with United States government support awarded by the National Institute of Environmental Health Sciences-MFBSC, Contract # ES05705, NIH-National Institute of Neurological Disorders and Stroke, Contract # NS36998, and National Institute of General Medicine, Contract # GM 57505. The United States has certain rights in this invention.BIBLIOGRAPHY [0003] Complete bibliographic citations of the references referred to herein by the first author's last name in parentheses can be found in the Bibliography section, immediately preceding the claims. FIELD OF THE INVENTION [0004] The invention relates to the field of biochemical assays and reagents. More specifically, this invention relates to fluorescent proteins and to methods for...

AI Technical Summary

Benefits of technology

[0012] The invention, which is defined by the claims set out at the end of this disclosure, is intended to solve at least some of the problems noted above. The invention provides improved fluorescent proteins with enhanced properties e.g., substantially enhanced fluorescence and reduced toxicity. The improved fluorescent proteins are useful in research and can be used, e.g., to determine or detect gene expression, e.g., up- or down-regulation, to monitor promoter activity, to allow longer term monitoring, and to localize proteins.

Problems solved by technology

However, factors other than fluorescence color and intensity affect the utility of a protein in research.

The stability of many fluorescent proteins makes them undesirable reporters to use if one seeks to determine short term or repetitive events.

Moreover, accumulated protein can be toxic to some mammalian cells.

Although the inventors do not wish to be limited to a single explanation of the toxicity of GFP from Aequorea, it is believed that this is probably due to free radical (H2O2) formation which occurs in a 1:1 stoichiometry with GFP production, making high levels of GFP expression particularly toxic.

This results in loss of fluorescence of the fluorophore.

This process is usually reversible but can limit the usefulness of GFP expression, e.g. by reducing time available to photograph specimens.

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